Intradermal Immunization of SARS-CoV-2 Original Strain Trimeric Spike Protein Associated to CpG and AddaS03 Adjuvants, but Not MPL, Provide Strong Humoral and Cellular Response in Mice.

Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.

Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

MPLA and AddaVax® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis.

There is so far no vaccine approved for human leishmaniasis, mainly because of the lack of appropriate adjuvants. This study aimed to evaluate in mice the capacity of a mixture of monophosphoryl lipid A (MPLA) and AddaVax® adjuvants in enhancing the efficacy of a Leishvacin®-like vaccine comprised of Leishmania amazonensis whole antigens (LaAg). For that, mice were immunized with LaAg plus MPLA/AddaVax® by the intramuscular route (i.m.) prior to challenge with 2 × 105 and 2 × 106 living parasites. Immunization with LaAg alone reduced the lesion growth of the 2 × 105-challenged mice only in the peak of infection, but that was not accompanied by reduced parasite load, and thus not considered protective. Mice given a 2 × 106 -challenge were not protected by LaAg. The association of LaAg with MPLA/AddaVax® was able to enhance the cutaneous hypersensitivity response compared with LaAg alone. Despite this, there was no difference in proliferative cell response to antigen ex vivo. Moreover, regardless of the parasite challenge, association of LaAg with MPL/AddaVax® did not significantly enhance protection in comparison with LaAg alone. This work demonstrated that MPL/AddaVax® is not effective in improving the efficacy of i.m. LaAg vaccine against cutaneous leishmaniasis.

B-1 lymphocytes are able to produce IL-10, but is not pathogenic during Leishmania (Leishmania) amazonensis infection.

Over the years research has found an association between B lymphocytes and pathogenesis during Leishmania sp. infections. Recently we demonstrated that B-2 lymphocytes are the main producers of IL-10 during L. amazonensis infection, and that the disease severity in BALB/c mice was attributed to these IL-10-producing B-2 lymphocytes. Here, we aim to understand the role of peritoneal B-1 lymphocytes in the pathogenesis of L. amazonensis infection. We found that infection resulted in a decrease in the number of B-1a lymphocytes and increase in B-1b lymphocytes in the peritoneal cavity of WT BALB/c mice but not in B lymphocyte deficient mice (BALB/Xid) mice. In vitro interaction between B-1 lymphocytes and L. amazonensis showed that the amastigote form of the parasite was able to induce higher levels of IL-10 in B-1 lymphocytes derived from infected BALB/c mice than the promastigote. Moreover, B-1 lymphocytes derived from infected mice produced more IL-10 than B-1 lymphocytes derived from naïve mice under amastigote interaction. However, the repopulation of BALB/Xid mice with B-1 lymphocytes from WT BALB/c mice did not affect the lesion development. Together, these results suggest that although B-1 lymphocytes are able to produce IL-10 during in vitro interaction with L. amazonensis, they are not directly related to pathogenesis in vivo.

Characterization of Sv129 Mice as a Susceptible Model to Leishmania amazonensis.

Leishmaniasis is a complex of neglected diseases caused by parasites of the genus Leishmania, such as Leishmania (Leishmania) amazonensis, the ethiologic agent of diffuse cutaneous leishmaniasis in Brazil. In this work, we investigated a new experimental model of infection for L. amazonensis: the Sv129 mouse. First, we subcutaneously infected Sv129 mice with 2 × 105 or 2 × 106 L. amazonensis parasites of the Josefa strain. A progressive lesion developed for both inoculation doses, showing that Sv129 mice are susceptible, independent of parasite dose. We next investigated the mechanisms associated with the pathogenesis of infection. We did not observe an increase of frequency of interferon-gamma (IFN- γ)-producing CD4+ and CD8+ T cells, a phenotype similar to that seen in BALB/c mice. There was an increased of frequency and number of IL-17-producing γδ (gamma-delta) T cells in infected Sv129 mice compared to naïve SV129 and an increased frequency of this population compared to infected BALB/c mice. In addition, Sv129 mice presented high levels of both IgG1 and IgG2a, suggesting a mixed Th1 and Th2 response with a skew toward IgG1 production based on IgG1/IgG2a ratio. Susceptibility of the Sv129 mice was further confirmed with the use of another strain of L. amazonensis, LTB0016. In this work, we characterized the Sv129 mice as a new model of susceptibility to Leishmania amazonensis infection, during infection there was controlled IFN-γ production by CD4+ or CD8+ T cells and induced IL-17 production by γδ T cells.

The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy.

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.